Despite optimized therapeutic regimens, the 5-year survival rate for head and neck squamous cell carcinoma (HNSCC) remains around 50%. EGFR is a key component of a receptor tyrosine kinase (RTK) network that functions as a non-mutated ?driver? in HNSCC cell lines and is the target for the FDA-approved agent, cetuximab. Individually, HNSCC patients exhibit wide-ranging extent of response to cetuximab as well as ERBB family- targeted tyrosine kinase inhibitors (TKIs) such as afatinib. Importantly, even in combination with chemo- or radiotherapy, EGFR inhibitors fail to eliminate 100% of tumor cells. These therapy-resistant tumor cells, termed ?residual disease cells?, have been invoked as a reservoir from which lethal drug resistant cancers ultimately emerge to drive progression. In support, the extent of therapy-induced tumor shrinkage correlates with progression-free survival in some cancers. Thus, deepening the HNSCC response to targeted drugs through mechanism-based combinations of agents that precisely attack the residual disease state is predicted to extend progression-free and overall survival. At present, the biological mechanisms that underlie tumor cell persistence and the heterogeneity of therapy- induced tumor responses observed in HNSCC patients are ill-defined, highlighting a critical knowledge gap. Our preliminary data demonstrate that inhibition of the EGFR-MEK-MAPK axis with targeted EGFR or MEK inhibitors in human and murine HNSCC cell lines rapidly stimulates an innate immune response leading to induction of chemokines and cytokines that signal to immune cells in the tumor microenvironment (TME) as well as antigen presentation pathways (MHC class I). Importantly, variable induction of the effector T cell and NK cell chemoattractant, CXCL10, pro-tumorigenic cytokines, TGF? and IL6, and MHC class I are observed within a panel of HNSCC cell lines, supporting the idea that intrinsic heterogeneity within this EGFR/MEK inhibitor- induced innate immune response instructs direct, yet variable immune cell participation in the therapeutic response. We propose to interrogate the HNSCC residual disease state with patient specimens, syngeneic murine tumor models and human HNSCC cell lines as a means for delineating novel paracrine pathways mediating therapy-induced cancer cell-TME cross-talk. This proposal will test the hypothesis that rapid transcriptional reprogramming stimulated by targeted EGFR-MEK-MAPK axis inhibitors induces a variable spectrum of anti- and pro-tumorigenic chemokines and cytokines that communicate with the immune microenvironment, leading to its direct participation in the therapeutic response. Moreover, intrinsic heterogeneity among HNSCC patients in the overall anti- vs. pro-tumorigenic balance of this innate response contributes to the observed variability in the extent of the observed responses. Successful completion of the studies may highlight presently unappreciated immune pathways for rational targeting with mechanism-based drug combinations in the future. Aim 1: Characterize early EGFR/MEK inhibitor-induced reprogramming and immune cell content in patient and murine orthotopic HNSCC tumors and associate with therapeutic response. The studies will test the hypothesis that greater degree of therapeutic response is linked to increased effector T cell infiltration and decreased myeloid cell content as well as increased MHC class I expression. Aim 2: Murine HNSCC cell lines will be propagated orthotopically in immune-competent and -deficient hosts to test the direct contributions of specific immune cell populations to the EGFR and MEK inhibitor therapeutic response. Aim 3: Define the mechanism of the EGFR/MEK inhibitor-induced innate immune response in HNSCC cell lines and the test the role in the in vivo therapeutic response.